Different caveolin isoforms in the retina of melanoma malignum affected human eye.

Purpose Caveolin-1 has been identified in Müller and pigment cells of rodents, but the distribution of caveolin isoforms has not been studied in the human retina. Since there are no relevant data in humans, we aimed to study the distribution of caveolins in the human retina. Methods Our samples were derived from eyes affected by melanoma malignum choroideae that were enucleated. The distribution of the caveolins was examined by immunocytochemistry using isoform-specific antibodies. Results In this study we report on the presence of different caveolin isoforms in many cell types of the human retina. These isoforms were present in several regions and layers in the human retina. Centro-peripheral changes have been detected: the distribution altered following the radier direction. Conclusions This is the first demonstration of caveolin expression in the human retina. Our data suggest that caveolins play an important role in the function of retinal cells. Our observations refute previous assumptions that there is a shortage of caveolins in the retina. Since the retina contains a number of different neuronal and glial cell types, the caveolin expression of these cells can no longer be a matter of dispute.

ments. We chose retinas affected by melanoma malignum choroideae, a common reason for enucleation. The quantity and the distribution of the caveolin isoforms were obviously different in the human retina compared to other species. The caveolins were present in many regions and layers in the human retina. They were present in both neuronal and glial cell types, which suggests, that caveolins must play a role in the functions of these cells.

METHODS
Material: Three patients were involved in the study: two males (ages: 50, 57) and one female (age: 50). These patients were operated after the diagnosis of posterior uveal melanoma (melanoma malignum choroideae) in 2005 at the Department of Ophthalmology, University of Debrecen. The enucleation was made without prior treatment (brachytherapy was disqualified). Thus the retinas were not damaged due to any other treatment and stayed as intact as possible.
After the enucleation the eyes were included into histopathological study. The histological types according to Callendar's classification were: spindle A (male patient, age: 50), dominantly spindle B (female patient, age: 50), and dominantly epitheloid tumor (male patient, age: 57), respectively. All experiments involving human subjects were carried out according to the Helsinki Declaration and with the approval of the Human Studies Ethical Committee, University of Debrecen.
Preparation of the retina: Immediately after enucleation a dorsal stitch was made for orientation. A non-infiltrated part of the eye was removed for further investigation, the rest was sent to histopathology. The former section was subsequently placed in the fixative (4% paraformaldehyde, in 0.1 M phosphate buffer, pH 7.4), and incubated for 24 h at 4 °C. Thereafter the solution was replaced with 0.1 M phosphate buffered saline (PBS, pH 7.4), and rinsed for at least 24 h before further processing. The retina was carefully detached from the posterior eyecup. Extreme care was taken to obtain samples from the peripheral part of the retina, and from behind the ora serrata. These ciliary and iridic regions can not be separated from the ciliary body and the iris, so they were handled together. For frozen sections the retinas were incubated overnight in 30% sucrose (diluted in 0.1 M phosphate buffer), embedded in cryomatrix (ThermoShandon Pittsburgh, PA), and frozen in liquid nitrogen. Next, 10 µm thick radial sections were prepared on a ThermoShandon cryotome. For radial semithin sectioning three samples were taken from the human retina following a radial plane including the macular region (M), periphery (P), ciliary body (CB).
Immunocytochemistry: Sections of 10 µm thickness were cut on a cryostat and dried onto poly-L-lysine coated glass microscope slides at 37 °C. Sections were then soaked in PBS for 20 min. A blocking solution of, 1% bovine serum albumin (BSA) with 0.1% Triton X-100 was applied for 2 h. The primary antibody was applied at 4 °C overnight. Primary antibodies anti-caveolin-1 (polyclonal rabbit IgG, Transduction Laboratories, BD Biosciences, San Jose, CA), 1:100, anti-caveolin-2 (monoclonal mouse IgG, Transduction Laboratories), 1:200, and anti-caveolin-3 (monoclonal mouse IgG, Transduction Laboratories) 1:100, were diluted in 1% BSA at 4 °C overnight. Caveolin-1 and -3 anti-rabbit and anti-mouse Alexa 488 (Molecular Probes, Invitrogen, Carlsbad, CA) were used as secondary antibodies. Since caveolin-2 gave a weakly detectable signal with the previous method, we used biotinylated anti-mouse IgG, then Streptavidin Alexa 488 (Molecular Probes), 1:100, to intensify the signal. Even with this extra method, caveolin-2 still gave a weak signal. For visualization of the cytoskeleton, we used Alexa fluor 594-labeled phalloidin (Molecular Probes) to stain F-actin (1:100). Vectashield HardSet Mounting Medium (Vector Laboratories, Burlingame, CA) with DAPI (4',6-diamidino-2-phenylindole) was used to cover the slides. When DAPI binds to DNA, its fluorescence is strongly enhanced, with excitation wavelength 350 nm (nuclei appear blue). Control reactions were made using rabbit and mouse normal serum, non-specific primary antibodies, and by omitting the primary antibodies to preclude non-specific binding. The retinas were inspected on a Zeiss Axiophot microscope Thornwood, NY, using the appropriate filter set for the fluorescent antibodies. Micrographs were obtained using an Olympus DP50 camera. Fluorescent doubleor triple-labeled specimens were also inspected on a Radiance 2100 Multi Photon Imaging System coupled to a Nikon Eclipse E800 microscope using LaserSharp 2000. Both Adobe Photoshop 7.0 and Confocal Assistant were used for primary image processing.

Caveolin-1:
The bound antibody was present in all layers of the retina. The weakest staining could be found in the pigment epithelium (low). The autofluorescence of the pigment granules made assessment of the immunostaining density more difficult. However the immunoreactive deposits were significantly smaller than the pigment granules, so the formers could be clearly identified. In other layers the density was estimated to vary from low to very high. The distribution of immunolabeling exhibited a characteristic center-to-peripheral gradient, increasing from very low, to low, moderate, and high density (M, Figure 1A,D,F), reaching the maximum level (very high; P Figure 1B Table 1).
Caveolin-2: Caveolin-2 immunoreactivities (IRs) were found to be present from the outer nuclear layer (ONL) to the inner limiting membrane (ILM), showing very low, low and moderate densities (M, Figure 2A,D,F). However no IRs were detected at the peripheral part (P, Figure 2B,E) in the IPL, the layer of the optic nerve fibers and the ILM. In the epithelium of the ciliary body (CB, Figure 2C) no IRs could be seen. (Figure 2, Table 2).
Caveolin-3: In the macular region, caveolin-3 immunoreactivity was present from the INL to the ILM with very low, low and moderate densities (M, Figure 3A,D,F). At the periphery (P, Figure 3B,E,G) IRs also were in the layer of the ©2007 Molecular Vision Molecular Vision 2007; 13:881-6 <http://www.molvis.org/molvis/v13/a96/> 882 outer segments (low). Caveolin-3 signals of low and moderate densities were present in both layers of the ciliary body (CB, Figure 3C, Table 3). Table 1, Table 2, and Table 3 present a summary of immunostaining densities in various tissue samples. Differences among the various sites were obvious, and only semiquantitative scoring was used. In contrast to monoclonal antibodies polyclonal sera may exhibit a reactivity against multiple epitopes. Accordingly, to avoid any uncertainty, caveolin isoform immunocytochemistry was performed with utmost care using a wide range of controls. For the same reason, the distribution of caveolin-1 detected by a polyclonal antibody was studied separately from that of the other two isoforms.
ported, caveolin-1 was present in the majority of retinal layers in the rat retina [12]. Caveolin-1 is known to be expressed abundantly in adipocytes, endothelial cells, fibroblasts, and smooth muscle cells. The observations of Song et al. and others confirm, that caveolin-1 is also expressed in some neuronal and glial cell types. No data are available about the distribution of caveolin-1 in the human retina yet, but studies on mammalian retinas suggest, that the densities of different caveolin types seem to be much lower compared to our sections. Caveolin-1 was only observed in a few cell types [9][10][11] and layers [12], including the ganglion cell layer, IPL, OPL, and in the vascular endothelial cells of the rat retina. Our observations show, caveolin-1 is evenly distributed among the different layers of the human retina, including the epithelium of the ciliary body. Although each cell type was not identified with specific markers, it was still obvious, that caveolin-1 is expressed in many cell types, including pigment cells, outer segments of photoreceptors, ganglion cells, and probably also in all the other cell types as well. Interestingly, the density of caveolin-1 exhibited a characteristic center-to-peripheral variation; after an increase toward the mid-periphery it reached a maximum level and then started to decrease toward the ora serrata.   Region  M  P  CB  -------------PE  ---OS  ---OLM  --ONL  ++  +  OPL  ++  ++  INL  +  ++  IPL  +  -GC  ++  ++  ONF  +++  -ILM +++ - Caveolin-2 was found in the outer nuclear layer (ONL) and the inner limiting membrane (ILM), including the intervening layers, showing very low, low and moderate densities (M). Very low and low density was seen at the periphery (P), but no signals were observed at the most peripheral part (CB).

Caveolin-2:
In previous studies, caveolin-2 was described to be present only around blood vessels of the rat retina [10,12]. This correlates with the fact, that caveolin-2 is expressed in adipocytes, endothelial cells, fibroblasts, and smooth muscle cells. Our observations confirmed the following: caveolin-2 is clearly localized around the human blood vessels. Caveolin-2 is also expressed in many layers in the human retina from the ONL to the ILM, indicating its presence in neuronal cell types. It is worth mentioning, that the most peripheral part of the human retina also expressed caveolin-2. The latter part, the epithelium of the ciliary body, was not examined previously in rodents.
Caveolin-3: Until the present study, no studies examined the presence of caveolin-3 in the human retina. Caveolin-3 remains to be studied in rodent retinas. Traditionally, caveolin-3 has been thought to be muscle specific, however it has been detected in astroglial cells and vegetative ganglions [8]. It is also known that the expression of caveolin-3 is intense in the central nervous system in early embryonic stages in the chicken [13].
The distribution of caveolin-3 in the human retina was similar to caveolin-2: IRs were present from the ONL to the ILM. At the most peripheral regions, IR density was prominent. During retinal development, central portions of the retina develop first, and peripheral areas develop later [14][15][16]. Sup-©2007 Molecular Vision Molecular Vision 2007; 13:881-6 <http://www.molvis.org/molvis/v13/a96/>  Region  M  P  CB  --------------PE  --++  OS  -++  +++  OLM  --ONL  ++  ++  OPL  ++  ++  INL  +  ++  IPL  +++  ++  GC  ++  ++  ONF  +++  ++  ILM  +++   porting previous observations about chicken development [13], caveolin-3 gives the highest density in this special, two layered peripheral part, which represents an early developmental stage of the retina. Conclusion: The quantity and distribution of the caveolin isoforms in the human retina are different from that found in other animals. Caveolins are present in more regions and in more layers in the human retina than in other species. Although caveolin is a conservative protein, the amount and distribution of caveolin in different tissues may exhibit considerable changes across species. Further data are needed to map the species-specific distribution of the detectable isoforms.
Further investigations are needed to explain the role played by caveolins in the intact human retina and in that of other species. Our observations demonstrate that in contrast to previous beliefs, there is no shortage of caveolins in the retina. It is hoped this study will facilitate additional research into the tissue specificity of caveolins. Since the retina contains a number of different neuronal and glial cell types, the caveolin expression of these cells can no longer be a matter of dispute.